Extended Data Fig. 7: Validation of informatics-processing steps: CCF registration and quantification from segmentation.

a–c, To determine the precision of the registration process on which we rely here for quantification of signal by layer in the cortex, we manually delineated layers 1 to 6b, using background fluorescence in coronal STPT images, for n = 9 cortical areas (ACAd, ORBvl, AId, PERI, SSp-bfd, MOp, VISp, RSPd, and AUDp; see Supplementary Table 3) in n = 4 mice per region. We then quantified the percentage of voxels within each manually annotated layer that were assigned to all cortical layers following automated registration to the CCFv3. a, A confusion matrix show the mean percentage of overlapping voxel labels averaged across these areas (individual region data in Supplementary Table 7). b, c, Boxplots show the median and mean (indicated with +); whiskers show the minimum–maximum range for the percentage overlap for individual experiments (b) or cortical areas (c, coloured dots). Across these cortical areas, the average percentage overlap ranged from 86 to 96% of voxels appropriately registered for all layers, except for L6b, which was not included in subsequent layer quantifications. For some areas and layers, the precision was worse than others; for example, while 66% of voxels were appropriately assigned to L2/3 in ACAd, the remaining 34% were assigned to neighbouring L5. In ORBvl, only 51% of voxels were appropriately labelled for L6a. Note, however, that delineating layer 5 from L6a in ORBvl in coronal sections using just background fluorescence was very difficult even for experienced anatomists, so some of the imprecision may in fact come from the manual drawing. Even with these exceptions noted, in all cases a large majority of voxels were registered and assigned correctly. d, e, Frequency distributions of informatically derived quantification for manually verified true negative and positive targets. d, The numbers of log₁₀-transformed normalized projection values are plotted for all CC and TC targets manually verified as true negative (n = 24,272) or true positive (n = 12,921). Most true positive values were between log₁₀ = −4 and log₁₀ = 1. At log₁₀ = −1.5 (red arrow), 639 true negatives remained (2.6%), while 7,100 true positives were still included (54.9%), resulting in a false positive rate of 8.3% at this threshold level. e, Numbers of log₁₀-transformed normalized projection values plotted for all CC and TC targets manually verified as true negative (n = 15,789) or true positive (n = 4,503). At log₁₀ = −2.5 (red arrow), 362 true negatives remained (2.3%), while 3,335 true positives were still included (74.1%), resulting in a false positive rate of 9.8% at this threshold level.